Improvement of Erythropoietin N-glycan Branching and Sialylation by Overexpression of Glycosyltransferases By:

............................................................................................................................. ii Acknowledgements .......................................................................................................... iv List of Figures .................................................................................................................. vii List of Tables .................................................................................................................. viii Chapter 1: Introduction ................................................................................................... 1 1.1 Protein Glycosylation and Sialylation ................................................................................ 1 1.1.1 Complex N-glycan Branching .................................................................................... 2 1.1.2 Sialylation ................................................................................................................... 4 1.2 Importance of Glycosylation and Sialylation of Recombinant Proteins Produced in Chinese Hamster Ovary Cells .................................................................................................. 6 1.3 Erythropoietin as a Model Protein for Glycoengineering Study ..................................... 6 Chapter 2: Incorporation of ST6GAL1, GNTIV and GNTV into CHO-K1 cells .... 10 2.1 Enhancing the Glycosylation and Sialylation of Recombinant Glycoproteins ............ 10 2.1.1 Overexpression of Sialylation Pathway Enzymes ........................................................ 10 2.1.2 Overexpression of N-acetylglucosaminyltransferases ................................................. 11 2.2 Previous Work ................................................................................................................... 12 2.3 Materials and Methods ..................................................................................................... 13 2.3.1 Cell Lines ..................................................................................................................... 13 2.3.2 Transfection .................................................................................................................. 14 2.3.3 Isolation of Single Clones by Limiting Dilution .......................................................... 14 2.3.4 Reverse Transcription PCR .......................................................................................... 15 2.3.5 Protein Purification ...................................................................................................... 15 2.3.6 SDS-PADE ................................................................................................................... 16 2.3.7 Western Blot ................................................................................................................. 16 2.3.8 Lectin Blot .................................................................................................................... 17 2.4 Results and Discussion ...................................................................................................... 18 2.4.1 Selection of the Stable Clone of ChEPO-S .................................................................. 18 2.4.2 Selection of the Stable Clone of ChEPO-SG ............................................................... 19 2.4.3 Expression of ST6GAL1, GNTIV and GNTV on Transcription Level ....................... 23 2.4.4 Expression of ST6GAL1, GNTIV and GNTV on Translation Level .......................... 24 Chapter 3: Enhanced N-glycan Branching and Sialylation of Recombinant Human Erythropoietin ................................................................................................................. 26 3.1 Glycoengineering for the Development of Biosimilars ................................................... 26 3.2 Materials and Methods ..................................................................................................... 28 3.2.1 Cell Lines ..................................................................................................................... 28 3.2.2 Immunoaffinity Purification of EPO ............................................................................ 28 3.2.3 SDS-PAGE ................................................................................................................... 28 3.2.4 Western Blot ................................................................................................................. 28 3.2.5 Lectin Blot .................................................................................................................... 29 3.2.6 Sialic Acid Analysis by High Performance Liquid Chromatography .......................... 29 3.3 Results and Discussion ...................................................................................................... 29 3.3.1 Lectin Blot Analysis for Various Erythropoietin Producing Cell Lines ...................... 29 3.3.2 Recombinant Erythropoietin Purified by Ni-NTA and Detected by Lectins ............... 31 3.3.3 Sialic Acid Content of Recombinant Erythropoietin Determined by HPLC................ 33